Bottom Line:
The loss of mifSR had no effect on the antibiotic resistance profile.We confirmed that the mifSR mutants have functional dehydrogenase complex suggesting a possible defect in α-KG transport.These data clearly suggests that P. aeruginosa MifSR TCS is involved in sensing α-KG and regulating its transport and subsequent metabolism.

Affiliation: Department of Biological Sciences, College of Arts & Sciences, Florida International University, Miami, Florida, United States of America; Department of Molecular Microbiology and Infectious Diseases, Herbert Wertheim College of Medicine, Florida International University, Miami, Florida, United States of America.

ABSTRACTPseudomonas aeruginosa is a Gram-negative, metabolically versatile opportunistic pathogen that elaborates a multitude of virulence factors, and is extraordinarily resistant to a gamut of clinically significant antibiotics. This ability, in part, is mediated by two-component regulatory systems (TCS) that play a crucial role in modulating virulence mechanisms and metabolism. MifS (PA5512) and MifR (PA5511) form one such TCS implicated in biofilm formation. MifS is a sensor kinase whereas MifR belongs to the NtrC superfamily of transcriptional regulators that interact with RpoN (σ54). In this study we demonstrate that the mifS and mifR genes form a two-gene operon. The close proximity of mifSR operon to poxB (PA5514) encoding a ß-lactamase hinted at the role of MifSR TCS in regulating antibiotic resistance. To better understand this TCS, clean in-frame deletions were made in P. aeruginosa PAO1 creating PAO∆mifS, PAO∆mifR and PAO∆mifSR. The loss of mifSR had no effect on the antibiotic resistance profile. Phenotypic microarray (BioLOG) analyses of PAO∆mifS and PAO∆mifR revealed that these mutants were unable to utilize C5-dicarboxylate α-ketoglutarate (α-KG), a key tricarboxylic acid cycle intermediate. This finding was confirmed using growth analyses, and the defect can be rescued by mifR or mifSR expressed in trans. These mifSR mutants were able to utilize all the other TCA cycle intermediates (citrate, succinate, fumarate, oxaloacetate or malate) and sugars (glucose or sucrose) except α-KG as the sole carbon source. We confirmed that the mifSR mutants have functional dehydrogenase complex suggesting a possible defect in α-KG transport. The inability of the mutants to utilize α-KG was rescued by expressing PA5530, encoding C5-dicarboxylate transporter, under a regulatable promoter. In addition, we demonstrate that besides MifSR and PA5530, α-KG utilization requires functional RpoN. These data clearly suggests that P. aeruginosa MifSR TCS is involved in sensing α-KG and regulating its transport and subsequent metabolism.

pone.0129629.g009: Growth curves in presence of α-KG in combination with succinate and citrate.To determine if α-KG is toxic to the cells, wild-type PAO1 and mifSR mutants were grown in the presence of α-KG in combination with succinate (A) and citrate (B) at 30 mM each. In comparison to the wild-type PAO1, mifSR mutants shared a similar exponential phase but reached stationary phase earlier, suggesting that it has depleted usable C-source. This suggests that PAO1 can efficiently utilize excess carbon source in the form of α-KG contributing to its increased growth.

Mentions:
α-KG is a key TCA cycle intermediate (Fig 8) and plays an important role in regulating carbon and nitrogen metabolism [44]. It has been previously shown that P. aeruginosa preferentially utilizes TCA cycle intermediates as a carbon source over other compounds [20,21,45]. To test if the growth defect exhibited by the loss of mifS and mifR is restricted to α-KG utilization, the mutants and the complementing strains were grown in the presence of TCA cycle intermediates citrate, succinate, fumarate, malate and oxaloacetate at 30 mM each. No difference in growth was observed between wild type PAO1 and its isogenic mutants in the presence of other TCA cycle intermediates except for α-KG (Table 1). This is not surprising as P. aeruginosa can use the glyoxylate shunt pathway to bypass the need for α-KG (Fig 8) [46]. Furthermore, no difference in the growth profile of the wild type PAO1 and mifSR mutants was observed when grown in the presence of sugars, glucose and sucrose (30 mM each) (Data not shown). To reconfirm that the presence of α-KG is not toxic, the cells were grown in the presence of citrate and succinate combined in equal concentration with α-KG. The mutants and the wild type shared similar early exponential growth (Fig 9). However, the mutants reached stationary phase earlier as compared to the parent strain PAO1. This suggests that the presence of excess carbon source in the form of α-KG further contributes to the growth of PAO1. These analyses indicate that mifSR mutants are only defective in α-KG utilization.

pone.0129629.g009: Growth curves in presence of α-KG in combination with succinate and citrate.To determine if α-KG is toxic to the cells, wild-type PAO1 and mifSR mutants were grown in the presence of α-KG in combination with succinate (A) and citrate (B) at 30 mM each. In comparison to the wild-type PAO1, mifSR mutants shared a similar exponential phase but reached stationary phase earlier, suggesting that it has depleted usable C-source. This suggests that PAO1 can efficiently utilize excess carbon source in the form of α-KG contributing to its increased growth.

Mentions:
α-KG is a key TCA cycle intermediate (Fig 8) and plays an important role in regulating carbon and nitrogen metabolism [44]. It has been previously shown that P. aeruginosa preferentially utilizes TCA cycle intermediates as a carbon source over other compounds [20,21,45]. To test if the growth defect exhibited by the loss of mifS and mifR is restricted to α-KG utilization, the mutants and the complementing strains were grown in the presence of TCA cycle intermediates citrate, succinate, fumarate, malate and oxaloacetate at 30 mM each. No difference in growth was observed between wild type PAO1 and its isogenic mutants in the presence of other TCA cycle intermediates except for α-KG (Table 1). This is not surprising as P. aeruginosa can use the glyoxylate shunt pathway to bypass the need for α-KG (Fig 8) [46]. Furthermore, no difference in the growth profile of the wild type PAO1 and mifSR mutants was observed when grown in the presence of sugars, glucose and sucrose (30 mM each) (Data not shown). To reconfirm that the presence of α-KG is not toxic, the cells were grown in the presence of citrate and succinate combined in equal concentration with α-KG. The mutants and the wild type shared similar early exponential growth (Fig 9). However, the mutants reached stationary phase earlier as compared to the parent strain PAO1. This suggests that the presence of excess carbon source in the form of α-KG further contributes to the growth of PAO1. These analyses indicate that mifSR mutants are only defective in α-KG utilization.

Bottom Line:
The loss of mifSR had no effect on the antibiotic resistance profile.We confirmed that the mifSR mutants have functional dehydrogenase complex suggesting a possible defect in α-KG transport.These data clearly suggests that P. aeruginosa MifSR TCS is involved in sensing α-KG and regulating its transport and subsequent metabolism.

Affiliation:
Department of Biological Sciences, College of Arts & Sciences, Florida International University, Miami, Florida, United States of America; Department of Molecular Microbiology and Infectious Diseases, Herbert Wertheim College of Medicine, Florida International University, Miami, Florida, United States of America.

ABSTRACTPseudomonas aeruginosa is a Gram-negative, metabolically versatile opportunistic pathogen that elaborates a multitude of virulence factors, and is extraordinarily resistant to a gamut of clinically significant antibiotics. This ability, in part, is mediated by two-component regulatory systems (TCS) that play a crucial role in modulating virulence mechanisms and metabolism. MifS (PA5512) and MifR (PA5511) form one such TCS implicated in biofilm formation. MifS is a sensor kinase whereas MifR belongs to the NtrC superfamily of transcriptional regulators that interact with RpoN (σ54). In this study we demonstrate that the mifS and mifR genes form a two-gene operon. The close proximity of mifSR operon to poxB (PA5514) encoding a ß-lactamase hinted at the role of MifSR TCS in regulating antibiotic resistance. To better understand this TCS, clean in-frame deletions were made in P. aeruginosa PAO1 creating PAO∆mifS, PAO∆mifR and PAO∆mifSR. The loss of mifSR had no effect on the antibiotic resistance profile. Phenotypic microarray (BioLOG) analyses of PAO∆mifS and PAO∆mifR revealed that these mutants were unable to utilize C5-dicarboxylate α-ketoglutarate (α-KG), a key tricarboxylic acid cycle intermediate. This finding was confirmed using growth analyses, and the defect can be rescued by mifR or mifSR expressed in trans. These mifSR mutants were able to utilize all the other TCA cycle intermediates (citrate, succinate, fumarate, oxaloacetate or malate) and sugars (glucose or sucrose) except α-KG as the sole carbon source. We confirmed that the mifSR mutants have functional dehydrogenase complex suggesting a possible defect in α-KG transport. The inability of the mutants to utilize α-KG was rescued by expressing PA5530, encoding C5-dicarboxylate transporter, under a regulatable promoter. In addition, we demonstrate that besides MifSR and PA5530, α-KG utilization requires functional RpoN. These data clearly suggests that P. aeruginosa MifSR TCS is involved in sensing α-KG and regulating its transport and subsequent metabolism.